Sex determination and larval sexual interaction in Bonellia viridis Rolando (Echiura : Bonelliidae)

Sex determination in the echiuran Bonellia viridis Rolando has classically been regarded as depending primarily on the environment of the newly settled larvae. The majority of the sexually undifferentiated larvae settling on an adult female become males; the larvae which settle away from the adult female become females in most cases. Previous work on this problem is reviewed. The behaviour, including the time-course of settlement, and the development of the indifferent larvae of B. viridis in the presence and absence of an adult female is described. Evidence is provided that even in the absence of adult females there is an interaction between newly settled larvae such that up to 20% of larvae become attached to one another in pairs with masculinization of one partner, the other developing into a female. Using larvae cultured singly, it was shown that crude extracts of adult female proboscis and trunk body-wall and the pigmented secretion of an irritated female masculinize indifferent larvae, the vast majority of which would have developed into females in pure sea water. Solutions of the purified integumentary pigment, bonellin, gave inconsistent results. Our experiments prove conclusively that sex determination is metagamic, i.e., not fixed at fertilization but is the result of an interaction between genetic and environmental factors, in ~83% of all larvae. The main environmental factor is a substance produced by the female. The other 17% are larvae whose sex is determined exclusively by their genetic make-up. These are syngamic males, females and intersexes. The problem of indefinitely undifferentiated larvae is discussed.peer-reviewe

Compartmentation of glycogen metabolism revealed from 13C isotopologue distributions

Background: Stable isotope tracers are used to assess metabolic flux profiles in living cells. The existing methods of measurement average out the isotopic isomer distribution in metabolites throughout the cell, whereas the knowledge of compartmental organization of analyzed pathways is crucial for the evaluation of true fluxes. That is why we accepted a challenge to create a software tool that allows deciphering the compartmentation of metabolites based on the analysis of average isotopic isomer distribution. Results: The software Isodyn, which simulates the dynamics of isotopic isomer distribution in central metabolic pathways, was supplemented by algorithms facilitating the transition between various analyzed metabolic schemes, and by the tools for model discrimination. It simulated 13C isotope distributions in glucose, lactate, glutamate and glycogen, measured by mass spectrometry after incubation of hepatocytes in the presence of only labeled glucose or glucose and lactate together (with label either in glucose or lactate). The simulations assumed either a single intracellular hexose phosphate pool, or also channeling of hexose phosphates resulting in a different isotopic composition of glycogen. Model discrimination test was applied to check the consistency of both models with experimental data. Metabolic flux profiles, evaluated with the accepted model that assumes channeling, revealed the range of changes in metabolic fluxes in liver cells. Conclusions: The analysis of compartmentation of metabolic networks based on the measured 13C distribution was included in Isodyn as a routine procedure. The advantage of this implementation is that, being a part of evaluation of metabolic fluxes, it does not require additional experiments to study metabolic compartmentation. The analysis of experimental data revealed that the distribution of measured 13C-labeled glucose metabolites is inconsistent with the idea of perfect mixing of hexose phosphates in cytosol. In contrast, the observed distribution indicates the presence of a separate pool of hexose phosphates that is channeled towards glycogen synthesis

Structure based inhibitor design targeting glycogen phosphorylase b. Virtual screening, synthesis, biochemical and biological assessment of novel N-acyl-β-d-glucopyranosylamines

Glycogen phosphorylase (GP) is a validated target for the development of new type 2 diabetes treatments. Exploiting the Zinc docking database, we report the in silico screening of 1888 β- D-glucopyranose-NH-CO-R putative GP inhibitors differing only in their R groups. CombiGlide and GOLD docking programs with different scoring functions were employed with the best performing methods combined in a “consensus scoring” approach to ranking of ligand binding affinities for the active site. Six selected candidates from the screening were then synthesized and their inhibitory potency was assessed both in vitro and ex vivo. Their inhibition constants’ values, in vitro, ranged from 5 to 377 µM while two of them were effective at causing inactivation of GP in rat hepatocytes at low µM concentrations. The crystal structures of GP in complex with the inhibitors were defined and provided the structural basis for their inhibitory potency and data for further structure based design of more potent inhibitors

Metformin lowers Glucose 6-phosphate in hepatocytes by activation of glycolysis downstream of glucose phosphorylation

The chronic effects of metformin on liver gluconeogenesis involve repression of the G6pc gene, which is regulated by the carbohydrate-response element-binding protein through raised cellular intermediates of glucose metabolism. In this study we determined the candidate mechanisms by which metformin lowers glucose 6-phosphate (G6P) in mouse and rat hepatocytes challenged with high glucose or gluconeogenic precursors. Cell metformin loads in the therapeutic range lowered cell G6P but not ATP and decreased G6pc mRNA at high glucose. The G6P lowering by metformin was mimicked by a complex 1 inhibitor (rotenone) and an uncoupler (dinitrophenol) and by overexpression of mGPDH, which lowers glycerol 3-phosphate and G6P and also mimics the G6pc repression by metformin. In contrast, direct allosteric activators of AMPK (A-769662, 991, and C-13) had opposite effects from metformin on glycolysis, gluconeogenesis, and cell G6P. The G6P lowering by metformin, which also occurs in hepatocytes from AMPK knockout mice, is best explained by allosteric regulation of phosphofructokinase-1 and/or fructose bisphosphatase-1, as supported by increased metabolism of [3-3H]glucose relative to [2-3H]glucose; by an increase in the lactate m2/m1 isotopolog ratio from [1,2-13C2]glucose; by lowering of glycerol 3-phosphate an allosteric inhibitor of phosphofructokinase-1; and by marked G6P elevation by selective inhibition of phosphofructokinase-1; but not by a more reduced cytoplasmic NADH/NAD redox state. We conclude that therapeutically relevant doses of metformin lower G6P in hepatocytes challenged with high glucose by stimulation of glycolysis by an AMP-activated protein kinase-independent mechanism through changes in allosteric effectors of phosphofructokinase-1 and fructose bisphosphatase-1, including AMP, Pi, and glycerol 3-phosphate

Identification of C-β-d-Glucopyranosyl Azole-Type Inhibitors of Glycogen Phosphorylase That Reduce Glycogenolysis in Hepatocytes: In Silico Design, Synthesis, in Vitro Kinetics, and ex Vivo Studies

Several C-β-d-glucopyranosyl azoles have recently been uncovered as among the most potent glycogen phosphorylase (GP) catalytic site inhibitors discovered to date. Toward further exploring their translational potential, ex vivo experiments have been performed for their effectiveness in reduction of glycogenolysis in hepatocytes. New compounds for these experiments were predicted in silico where, for the first time, effective ranking of GP catalytic site inhibitor potencies using the molecular mechanics-generalized Born surface area (MM-GBSA) method has been demonstrated. For a congeneric training set of 27 ligands, excellent statistics in terms of Pearson (RP) and Spearman (RS) correlations (both 0.98), predictive index (PI = 0.99), and area under the receiver operating characteristic curve (AU-ROC = 0.99) for predicted versus experimental binding affinities were obtained, with ligand tautomeric/ionization states additionally considered using density functional theory (DFT). Seven 2-aryl-4(5)-(β-d-glucopyranosyl)-imidazoles and 2-aryl-4-(β-d-glucopyranosyl)-thiazoles were subsequently synthesized, and kinetics experiments against rabbit muscle GPb revealed new potent inhibitors with best Ki values in the low micromolar range (5c = 1.97 μM; 13b = 4.58 μM). Ten C-β-d-glucopyranosyl azoles were then tested ex vivo in mouse primary hepatocytes. Four of these (5a–c and 9d) demonstrated significant reduction of glucagon stimulated glycogenolysis (IC50 = 30–60 μM). Structural and predicted physicochemical properties associated with their effectiveness were analyzed with permeability related parameters identified as crucial factors. The most effective ligand series 5 contained an imidazole ring, and the calculated pKa (Epik: 6.2; Jaguar 5.5) for protonated imidazole suggests that cellular permeation through the neutral state is favored, while within the cell, there is predicted more favorable binding to GP in the protonated form

Aspects of the metabolism of the lactating rat

The effects of alterations in endocrine and nutritional state on lipogenesis in vivo in mammary gland, liver, white and brown adipose tissue (WAT,BAT) of the lactating rat were investigated. During lactation, lipogenesis was increased in liver, decreased in BAT and unchanged or decreased in WAT; the mammary gland was the major site of lipogenesis. Insulin administration did not increase lipogenesis in the gland any further but it increased it in liver, WAT and BAT. Removal of the pups or suppression of prolactin secretion increased the plasma insulin concentration and lipogenesis in liver, WAT and BAT. There may be a reciprocal relationship between plasma prolactin and insulin, and the hypoinsulinaemia of lactation may be important in restricting lipogenesis in liver, WAT and BAT to promote the direction of glucose to the gland. An oral load of glucose increased lipogenesis in vivo 2-fold in liver of virgin and lactating rats and 8-10- fold in BAT of virgin rats but had no effect on BAT of lactating rats. BAT slices from lactating rats showed decreased rates of lipogenesis in vitro. An oral load of medium- or long-chain triacylglycerol inhibited lipogenesis in mammary gland in vivo (82-89%) This inhibition was reversed when the acini were incubated in vitro. Insulin administration reversed partially the inhibition of lipogenesis in vivo. Long-chain triacylglycerol inhibited hepatic lipogenesis in vivo but medium-chain triacylglycerol increased it 2-fold. Lactating rats fed a high-energy diet had a decreased rate of mammary gland lipogenesis in vivo (60-80%). Glucose uptake and lipogenesis by the isolated acini in vitro correlated with the depressed lipogenesis in vivo. Insulin in vitro reversed glucose uptake and lipogenesis to values approaching those of controls fed on chow diet. In lactating rats the high-energy diet did not affect hepatic and WAT lipogenesis but it increased BAT lipogenesis. In virgin rats the high-energy diet increased WAT lipogenesis but it decreased hepatic and BAT lipogenesis. It is concluded that in lactating rats the highenergy diet results in a decrease in sensitivity of the mammary gland to insulin and in increased partitioning of carbohydrate to BAT while in virgin rats excess carbohydrate is partitioned to lipogenesis in WAT.</p